1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology. Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Of these, the phosphoramidite approach has become the most popular for most applications. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. The phosphoramidite approach has been used to synthesize oligonucleotides having a variety of modified internucleoside linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Solid phase synthesis of oligonucleotides by the phosphoramidite approach can be varied for different applications, but ordinarily involves the same generalized protocol. Briefly, this approach comprises anchoring the 3xe2x80x2-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3xe2x80x2 phosphoramidite group of the incoming nucleoside and the 5xe2x80x2 hydroxyl group of the 5xe2x80x2-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 umol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4: 185 (1994). Several modifications of the standard phosphoramidite methods have already been reported to facilitate the synthesis and isolation of oligonucleotides. See e.g., Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50: 9255 (1994); Theisen et al., Nucleosides and Nucleotides 12: 43 (1994); and Iyer et al., Nucleosides and Nucleotides 14: 1349 (1995) (Kuijpers et al., Nucl. Acids Res. 18: 5197 (1990); and Reddy et al., Tetrahedron Lett. 35: 4311 (1994).
A major limiting factor for cost efficient synthesis of oligonucleotides is the time and cost required to make and purify the monomeric nucleoside phosphoramidites. Bodepudi et al., Chem. Res. Toxicol. 5: 608-617, discloses that the preparation of phosphoramidites from 2xe2x80x2-deoxy-7,8-dihydro-8-oxoguanosine and 2xe2x80x2-deoxy-7,8-dihydro-8-oxoadenosine according to the standard procedure results in extensive decomposition of the phosphoramidites during purification due to their instability and sensitivity to water. One potential approach to overcome these problems is to generate the phosphoramidite in situ as the oligonucleotide synthesis process is being carried out. Unfortunately, the numerous attempts at this approach have been disappointing. Moore and Beaucage, J. Org. Chem. 50: 2019-2025 (1985) teaches in situ preparation of phosphoramidites by reacting deoxyribonucleosides with bis-(pyrrolidino)methoxyphosphine activated by 4,5-dichloroimidazole in 1-methyl-2-pyrrolidinone. However, this method was limited by poor chemoselectivity, with about 8-10% (3xe2x80x2-3xe2x80x2)-dinucleoside methyl phosphite triester being formed as a by-product. Barone et al., Nucleic Acids Res. 12: 4051-4061 (1984) and Lee and Moon, Chem. Lett. 1229-1232 (1984) disclose better chemoselectivity in preparation of phosphoramidites in situ, by reacting deoxyribonucleosides with bis-(N,N,-dialkylamino)alkoxyphosphines and 1H-tetrazole or its N,N-diisopropylammonium salt. Unfortunately, the tetrazole-N,N-diisopropylammonium salt, either added or generated in situ may form precipitates inside the synthesizer. Helinski et al., Tetrahedron Lett. 32: 4981-4984 (1991) and 34: 6451-6454 (1993) disclose selective activation of bifunctional phosphitylating reagents containing a p-nitrophenoxy group. However, this methodology is not adaptable in current phosphoramidite approaches because the p-nitrophenoxy group has to be activated by using a strong base. Finally, Fourrey et al., Tetrahedron Lett. 22: 729-732 (1981) and Cao et al., Tetrahedron Lett. 24: 1019-1020 (1983) disclose, as reactive bifunctional phosphitylating agents, phosphorodichlorite and the corresponding ditetrazolite and ditriazolite. Unfortunately, the application of these agents to the synthesis of oligonucleotides is generally problematic, because of their extremely high reactivity and poor chemoselectivity.
There is, therefore, a need for new bifunctional phosphitylating reagents and their application in in situ preparation of 5xe2x80x2-protected nucleoside phosphoramidites and synthesis of oligonucleotides without prior purification of the nucleoside phosphoramidites. Ideally, such reagents should react quickly with nucleosides under neutral or weakly basic conditions, without an additional activation step, should generate chemoselectively the corresponding nucleoside phosphoramidites in situ, and should be relatively stable and easy to handle.
The invention provides novel bifunctional phosphitylating reagents and their application in in situ preparation of 5xe2x80x2-protected nucleoside phosphoramidites and synthesis of oligonucleotides. Bifunctional phosphitylating reagents according to the invention react quickly with nucleosides under neutral or weakly basic conditions, without an additional activation step. In addition, the bifunctional phosphitylating reagents according to the invention generate chemoselectively the corresponding nucleoside phosphoramidites in situ, without the need to purify the nucleoside phosphoramidites before using them in oligonucleotide synthesis. Finally, the bifunctional phosphitylating reagents according to the invention are relatively stable and easy to handle.
In a first aspect, the invention provides bifunctional phosphitylating reagents which are useful for in situ preparation of 5xe2x80x2-protected nucleoside phosphoramidites and synthesis of oligonucleotides. Bifunctional phosphitylating reagents according to the invention have the general structure (I): 
wherein the right-most O, C, or S is the point of attachment to phosphorous; 
wherein the left-most N is the point of attachment to phosphorous; 
wherein the left-most N is the point of attachment to phosphorous. Bifunctional phosphitylating reagents according to the invention react in the presence of a secondary or tertiary amine with 5xe2x80x2-protected nucleosides to chemoselectively produce 5xe2x80x2-protected nucleoside-3xe2x80x2-phosphoramidites.
In a second aspect, the invention provides a process for generating 5xe2x80x2-protected nucleoside phosphoramidites in situ, without producing a precipitate and without requiring purification of the nucleoside phosphoramidites prior to their use in oligonucleotide synthesis. In the process according to this aspect of the invention, bifunctional phosphitylating reagents according to the invention are reacted with 5xe2x80x2-protected nucleosides in the presence of a secondary or tertiary amine to produce a 5xe2x80x2-protected nucleoside phosphoramidite.
In a third aspect, the invention provides an improved process for synthesizing an oligonucleotide. In the process according to this aspect of the invention, the improvement comprises the step of generating the nucleoside phosphoramidite in situ, rather than adding purified nucleoside phosphoramidites at the appropriate point in a conventional oligonucleotide synthesis procedure.
The reagents and processes according to the invention are useful for producing a wide variety of unmodified or chemically modified oligonucleotide compounds, or radiolabeled oligonucleotide compounds, all of which are referred to herein generally as xe2x80x9coligonucleotidesxe2x80x9d. The reagents and processes according to the invention can be used or practiced on a scale ranging from a small laboratory scale to a large commercial scale.